Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples
Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spec...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2015-06-01
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| Series: | EuPA Open Proteomics |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2212968515000070 |
| _version_ | 1811217774864760832 |
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| author | Christina Looße Sara Galozzi Linde Debor Mattijs K. Julsing Bruno Bühler Andreas Schmid Katalin Barkovits Thorsten Müller Katrin Marcus |
| author_facet | Christina Looße Sara Galozzi Linde Debor Mattijs K. Julsing Bruno Bühler Andreas Schmid Katalin Barkovits Thorsten Müller Katrin Marcus |
| author_sort | Christina Looße |
| collection | DOAJ |
| description | Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures. |
| first_indexed | 2024-04-12T07:00:01Z |
| format | Article |
| id | doaj.art-15244b88a1444abea96386e7f754c1b4 |
| institution | Directory Open Access Journal |
| issn | 2212-9685 |
| language | English |
| last_indexed | 2024-04-12T07:00:01Z |
| publishDate | 2015-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | EuPA Open Proteomics |
| spelling | doaj.art-15244b88a1444abea96386e7f754c1b42022-12-22T03:43:01ZengElsevierEuPA Open Proteomics2212-96852015-06-017C202610.1016/j.euprot.2015.03.001Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samplesChristina Looße0Sara Galozzi1Linde Debor2Mattijs K. Julsing3Bruno Bühler4Andreas Schmid5Katalin Barkovits6Thorsten Müller7Katrin Marcus8Medizinisches Proteom-Center, Ruhr-University Bochum, D-44801 Bochum, GermanyMedizinisches Proteom-Center, Ruhr-University Bochum, D-44801 Bochum, GermanyLaboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, GermanyLaboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, GermanyLaboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, GermanyLaboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, GermanyMedizinisches Proteom-Center, Ruhr-University Bochum, D-44801 Bochum, GermanyMedizinisches Proteom-Center, Ruhr-University Bochum, D-44801 Bochum, GermanyMedizinisches Proteom-Center, Ruhr-University Bochum, D-44801 Bochum, GermanyRelative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.http://www.sciencedirect.com/science/article/pii/S2212968515000070Direct infusionQuantificationSingle ion monitoring (SIM)Q ExactiveCytochrome P450 (CYP) |
| spellingShingle | Christina Looße Sara Galozzi Linde Debor Mattijs K. Julsing Bruno Bühler Andreas Schmid Katalin Barkovits Thorsten Müller Katrin Marcus Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples EuPA Open Proteomics Direct infusion Quantification Single ion monitoring (SIM) Q Exactive Cytochrome P450 (CYP) |
| title | Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples |
| title_full | Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples |
| title_fullStr | Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples |
| title_full_unstemmed | Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples |
| title_short | Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples |
| title_sort | direct infusion sim as fast and robust method for absolute protein quantification in complex samples |
| topic | Direct infusion Quantification Single ion monitoring (SIM) Q Exactive Cytochrome P450 (CYP) |
| url | http://www.sciencedirect.com/science/article/pii/S2212968515000070 |
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